The broad goal of this research program is to advance our understanding of the cell physiology of cultured transporting epithelia so that the advantages offered by tissue culture technology can be exploited more effectively to probe transport mechanisms in specific epithelial cell types at the cellular and subcellular levels. The proposed studies will center on cells derived from the mouse medullary thick ascending limb and the rabbit cortical collecting tubule. The major questions that will be investigated are: (1) In what ways do the structural and transport characteristics of cultured renal epithelial cells correspond to or differ from those of intact nephrons? On the basis of prior studies with epithelia and a variety of other specialized cell types it is expected that differentiated epithelial morphological and transport properties will change in culture. This leads to the second major question: (2) What are the biochemical and metabolic alterations in renal cells which accompany the decay in transport phenotype? With basic information about the processes taking place in dedifferentiating cultured epithelia we will be in a better position to nonempirically approach the final question of: (3) What modifications in our current methods for maintaining transporting epithelial cells in culture will lead to stable cell populations which continue to express their in situ phenotype? No previous studies with mammalian transporting epithelia have dealt with these questions. To benefit fully from the potential advantages offered by cell culture systems for analyzing epithelial transport mechanisms (see Research Plan, Section B) the problem of phenotypic stability must be appreciated and resolved. If this is not done, the physiological significance of data generated from cell culture systems will always be open to question.